Melanoma: Metabolic Biomarkers of Response to Targeted Therapy Project Summary/Abstract Disseminated metastatic melanoma is initially treated with inhibitors of the V600E mutated BRAF kinase, a component of the MAPK signaling pathway that controls the replication of melanoma cells. More than half of melanoma patients express this mutation and are at least initially responsive to its inhibition. However, all pa- tients eventually become resistant to these inhibitors and have to be treated with alternate therapy, which con- sists primarily of immune checkpoint inhibitors. The goal of this project is to develop an imaging method that could monitor the effectiveness of BRAF kinase inhibitors and can promptly and accurately detect resistance to these agents. Our strategy for achieving this goal is to study the detailed biochemical mechanism of BRAF ki- nase signaling on the premise that a change in tumor metabolism is a quicker and more reliable indicator of the onset of resistance than a change in tumor volume, which can require weeks to months to become reliably manifest. We will use 13C MRS and liquid chromatography mass-spectrometry (LC-MS) to study the mecha- nism of BRAF metabolic inhibition, but these methods are not suitable for in vivo detection in humans ? 13C MRS is not sensitive enough and LC-MS is invasive. Therefore, our strategy is to identify suitable biomarkers of metabolic response that can be monitored by 1H MRS or MRI monitored chemical exchange saturation transfer (CEST), which is about 500 times more sensitive than 1H MRS but requires high magnetic field instru- ments operating at ? 7T. In contrast, 1H MRS can be monitored at 1.5T or 3T, for which instruments are avail- able at many more medical institutions. Our second objective is to delineate how the biomarkers of BRAF inhi- bition work in order to better appreciate their capabilities and limitations. Finally, preliminary data from our own lab and from others indicates that the onset of resistance to mutant BRAF inhibitors involves a transition of the tumor from dependence on aerobic glycolysis to substantially greater dependence on ox-phos and on glutami- nolysis. This has led to clinical trials of the use of inhibitors of ox-phos such as metformin and phenformin to delay the onset of resistance. As our third objective, we propose to test the feasibility of using CB-839, an in- hibitor of glutaminase to block the transition to glutamine-dependence as a method to inhibit the onset of BRAF resistance. Our Specific Aims are: Aim 1 will determine biochemical changes and effectiveness of MAPK pathway inhibition in murine and human models of melanoma. Aim 2 will elucidate substrate limitations on bio- chemical effects and biomarker response to changes in microenvironment. Aim 3 will validate the proposed biomarkers in an in vivo system where the treatment response is modified using a glutaminase inhibitor. Clinical Impact: This project will enable detection of melanoma response to targeted therapy by NMR meth- ods that have already been implemented in the clinic on conventional 1.5T and 3T instruments and on 7T in- struments that are becoming progressively more common. Metabolomic studies will expand our ability to quan- tify tumor metabolism in cells, in mouse and human models, and eventually in humans.